Project Summary Activity-dependent signaling pathways are crucial to synapse development and function, and defects in these pathways result in neurodegeneration, depression, among other developmental disorders. One such signaling pathway, the conserved bone morphogenetic protein (BMP) pathway, directs NMJ growth and neurotransmitter release at the Drosophila melanogaster neuromuscular junction (NMJ). Interestingly, tissue-specific rescue experiments have demonstrated that distinct pools of the BMP ligand Glass bottom boat (Gbb) control NMJ structure and function. While postsynaptic Gbb promotes overall NMJ growth, presynaptic Gbb promotes neurotransmission. Excitingly, through our discovery of the neuronal transmembrane protein Crimpy, we were able to identify the presynaptic pool of Gbb. We found that Crimpy delivers neuronal Gbb to dense core vesicles (DCVs) for activity-dependent release at the NMJ. Upon DCV exocytosis, both a Crimpy C-terminal fragment and Gbb are released from presynaptic terminals, defining an activity-dependent presynaptic signal. Our preliminary data show that mutants lacking a presynaptic Gbb signal exhibit fewer and larger neurotransmitter release sites, as well as display aberrant pre- and postsynaptic organization, via high- resolution STED and electron microscopy techniques at a relatively late stage of development. This argues that presynaptic Gbb is required to drive proper synapse organization. Remarkably, these defects strongly resemble those exhibited in mutants of the small GTPase Rab3, which is known to be involved DCV biogenesis and/or exocytosis. To reveal how presynaptic Gbb signaling impinges on synapse organization, I will first assess whether presynaptic Gbb regulates the localization of key synaptic structural components. Next, I will elucidate how presynaptic Gbb controls synaptic vesicle dynamics by performing electrophysiological techniques to investigate function and synaptic vesicle distribution in mutants lacking presynaptic Gbb. In addition, the synaptic defects observed at these late developmental stages beg the question of whether Gbb is only required for maintenance of synapses or if it is also required for initial synapse assembly. Presently, we are determining if presynaptic Gbb is an early synaptogenic cue by performing a live embryo dissection technique that was developed in our lab. I will also address key issues regarding the timing of presynaptic Gbb action. Lastly, in order to determine the role of the activity-dependent Gbb signal in directing synapse structure and function, I will test whether Rab3 and presynaptic Gbb function in a common pathway to regulate synapse architecture. Through these studies, we aim to define the role of the activity-dependent Gbb signal in synaptic assembly, maintenance, and function.